Mastercycler epgradient 5341 thermal cycler

HeLa-d1-eGFP cells were seeded 1 × 10⁵ cells per well in a 12-well plate and incubated for 24 h prior to siRNA treatment with siGFP-1 or negative control siRNA. Serial dilution of siRNA, lipoplex formulation and treatment were carried out as described above. Negative control siRNA was prepared at the same concentration as the highest siGFP dose. After 24 h lipoplex incubation, cells were washed with PBS, and RNA was extracted using GenElute Mammalian Total RNA Miniprep Kit according to the manufacturer’s protocol. Complementary DNA was obtained using SuperScript III First-Strand Synthesis System (Sigma) with random hexamer primers running on a MasterCycler EpGradient 5341 thermal cycler (Eppendorf AG, Hamburg, Germany). Real-Time qPCR was performed on a StepOnePlus Real-Time PCR System using MicroAmp Fast 0.1 mL 96-well Reaction Plates (Applied Biosystems, Foster City, CA, USA) and SYBR Green JumpStart Taq Readymix (Sigma) for the qPCR reactions. GAPDH was used as a housekeeping gene, data were analyzed with StepOne Software v.2.3, and d1-eGFP expression fold-change relative to the control sample was calculated using the ∆∆Ct method.